Bowman-Birk inhibitor mutants of soybean generated by CRISPR-Cas9 reveal drastic reductions in trypsin and chymotrypsin inhibitor activities

Authors: KIM, WON-SEOK - University Of Missouri; Gillman, Jason; KIM, SUNHYUNG - University Of Missouri; LIU, JUNQI - University Of Minnesota; JANGA, MADHUSUDHANA - Texas Tech University; STUPAR, ROBERT - University Of Minnesota; Krishnan, Hari

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/15/2024
Publication Date: 5/21/2024
Citation: Kim, W., Gillman, J.D., Kim, S., Liu, J., Janga, M.R., Stupar, R.M., Krishnan, H.B. 2024. Bowman-Birk inhibitor mutants of soybean generated by CRISPR-Cas9 reveal drastic reductions in trypsin and chymotrypsin inhibitor activities. International Journal of Molecular Sciences. 25(11). Article 5578. https://doi.org/10.3390/ijms25115578.
DOI: https://doi.org/10.3390/ijms25115578

Interpretive Summary: Soybean meal is often the first choice for swine and poultry feed mixtures due to a balanced amino acid profile. Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included into animal feed mixtures due to anti-nutritional compounds that interfere with digestion and effectively reduce animal weight gain. Soybean seeds contain Bowman-Birk inhibitors (BBi), which inactivate the animal digestive enzymes trypsin and chymotrypsin. Eliminating the BBi in soybean seeds will greatly improve the animal performance and enhance the value of soybean meal. In this study, we have utilized CRISPR/Cas9 gene editing technology to induce mutations in soybean seed BBi genes to decrease or eliminate their accumulation in seeds. Here, we report the successful creation of BBi mutants with substantial reductions in both trypsin and chymotrypsin inhibitor activities. The results of our study will benefit US soybean farmers by promoting the wider use of the newly developed BBi mutants in animal feed mixtures and for human food products, such as tofu and soymilk.

Technical Abstract: Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman–Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities.